PCR Lab Report PDF

Preview

Summary

PCR lab report ...

Description

Abbie-lyn Smith BIO- 181L F20 12/9/2020 Kylie Kramer

Application of Molecular Biology Techniques Introduction PCR is a repetitive cycle that involves three steps, this includes: denaturation, annealing and extension. Denaturation is done by increasing the temperature in order to break the bonds that bind the DNA double helix. Followed by the annealing process, which involves reducing the temperature to cause the primers to bind to specific sequences of the template DNA. The last part of PCR is the extension which involves increasing the temperature to an optimal temperature for DNA polymerase which will allow for the creation and addition of new strands of DNA. In this lab we are analyzing and amplifying the TPA 25 gene with and without the ALU repeat. First the DNA is extracted from each of the students in order to analyze it and amplify the genes. DNA extraction is used to isolate the DNA from the nucleus cells. PCR is used to amplify a defined, specified region of DNA by the usage of two designed, synthetic DNA oligonucleotides and is also used to make copies of the specified gene. After PCR is completed, Agarose Gel Electrophoresis is performed to analyze the results, this separates the DNA, RNA and proteins based on their fragments depending on their size and charge. Gel electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an electric current, and it

separates DNA fragments according to size. A standard, or DNA ladder, is typically included so that the size of the fragments in the PCR sample can be determined. PCR and gel electrophoresis produce enough copies of a DNA sequence that we can see or manipulate that region of DNA. Data

Analysis The results of this experiment showed that PCR does work. There were 12 student’s samples that were successfully amplified. 8 of the student’s samples were unsuccessful. The 12 samples produced 24 alleles. This is because there were 7 students that were homozygous with Alu, 3 students that were homozygous without and 2 students that were with and without Alu. The students homozygous with Alu resulted in 14 alleles, the students homozygous without Alu resulted in 6 alleles, and the students heterozygous with and without Alu made up for the last 4 alleles. The loading buffer used in agarose gel electrophoresis had 3 primary functions loading dye in order to keep the DNA from floating in the buffer solution, there two different speeds of dye, one that moves faster than DNA and one that moves slower so one can determine the relative position of the DNA. It also makes sure that the samples are kept in their original state based on their charge and size. A DNA ladder is also used in the agarose gel which is a solution

of different lengths of DNA molecules applied to the gel in order to determine the relative size of the DNA molecules. The bands on the gel represent if a person is positive or negative for the Alu elements. The classes DNA was amplified in order to determine if the DNA in the classes body was positive for the Alu element. Having the positive and negative control was to see if DNA was amplified, and to see if the DNA results had the Alu element present. The negative control had no DNA template, which would mean nothing was amplified. The positive control was being heterozygous for the Alu elements. This would be between homologous positive Alu and homologous negative Alu. This made two bands appear, and if only one showed up, then it was proven that the person either was positive or negative for the Alu elements. If no bands were to appear for a student, this would mean that their DNA was not amplified. Conclusion This lab was able to show the presence of Alu activity in an individual’s DNA. The TPA25 is a gene that was amplified for that purpose. This is because the gene was amplified with or without the Alu insert which was able to distinguish the samples of DNA that was with and without Alu. There are errors that could have happened in this experiment. Any inaccuracy with the DNA sample could have resulted from any of the samples being spilled or broken through the gel, or not remembering which sample was put into each slot of the DNA ladder.

References Areda, D., Boyles, R., Francis, G., & Hite, A. (2016). Laboratory manual for General Biology I. Retrieved 9 December 2020 from http://lc.gcumedia.com/bio181l/laboratory-manualfor-general-biology-i/v1.1/#/chapter/6 Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., Jackson, R., & Campbell, N. A. (2011). Campbell Biology. Retrieved 9 December 2020, from https://viewer.gcu.edu/24WWXP...